Calcium and calmodulin regulate diverse cellular processes in plants (Poovaiah and Reddy, CRC Crit. Rev. Plant Sci. 6:47-103, 1987, and CRC Crit. Rev. Plant Sci. 12:185-211, 1993; Roberts and Harmon, Annu. Rev. Plant Physiol. Plant Mol. Biold. 43:375-414, 1992; Gilroy and Trewavas, BioEssays 16:677-682, 1994). Transient changes in intracellular Ca.sup.2+ concentration can affect a number of physiological processes through the action of calmodulin (CaM), a ubiquitous Ca.sup.2+ -binding protein. Ca.sup.2+ /calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of Ca.sup.2+ -mediated signals (Veluthambi and Poovaiah, Science 223:167-169, 1984; Schulman, Curr. Opin. in Cell. Biol. 5:247-253, 1993). Extracellular and intracellular signals regulate the activity of protein kinases, either directly or through second messengers. These protein kinases in turn regulate the activity of their substrates by phosphorylation (Cohen, Trends Biochem. Sci. 17:408-413, 1992; Stone and Walker, Plant Physiol. 108:451-457, 1995).
In animals, Ca.sup.2+ /calmodulin-dependent protein kinases play a pivotal role in cellular regulation (Colbran and Soderling, Current Topics in Cell. Reg. 31:181-221, 1990; Hanson and Schulman, Annu. Rev. Biochem. 61:559-601, 1992; Mayford et al., Cell 81:891-904, 1995). Several types of CaM-dependent protein kinases (CaM kinases, phosphorylase kinase, and myosin light chain kinase) have been well characterized in mammalian systems (Fujisawa, BioEssays 12:27-29, 1990; Colbran and Soderling, Current Topics in Cell. Reg. 31:181-221, 1990; Klee, Neurochem. Res. 16:1059-1065, 1991; Mochizuki et al., J. Biol. Chem. 268:9143-9147, 1993).
Although little is known about Ca.sup.2+ /calmodulin-dependent protein kinases in plants (Poovaiah et al., in Progress in Plant Growth Regulation, Karssen et al., eds., Dordrecht, The Netherlands: Kluwer Academic Publishers, 1992, pp. 691-702; Watillon et al., Plant Physiol. 101:1381-1384, 1993), Ca.sup.2+ -dependent, calmodulin-independent protein kinases (CDPKs) have been identified (Harper et al., Science 252:951-954, 1991; Roberts and Harmon, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:375-414, 1992).
Male gametophyte formation in the anther is a complex developmental process involving many cellular events. During microsporogenesis, microsporocytes undergo meiosis to form tetrads of microspores that are surrounded by a callose wall composed of .beta.-1,3-glucan. The callose wall is subsequently degraded by callase, which is secreted by cells of the tapetum (Steiglitz, Dev. Biol. 57:87-97, 1977), a specialized anther tissue that produces a number of proteins and other substrates that aid in pollen development or become a component of the pollen outer wall (Paciani et al., Plant Syst. Evol. 149:155-185, 1985; Bedinger, Plant Cell 4:879-887, 1992; Mariani et al., Nature 347:737-741, 1990). The timing of callase secretion is critical for microspore development. Male sterility has been shown to result from premature or delayed appearance of callase (Worral et al., Plant Cell 4:759-771, 1992; Tsuchiya et al., Plant Cell Physiol. 36:487-494, 1995).
Induction of male sterility in plants can provide significant cost savings in hybrid plant production, enable production of hybrid plants where such production was previously difficult or impossible, and allow the production of plants with reduced pollen formation to reduced the tendency of such plants to elicit allergic reactions or to extend the life of flowers that senesce upon pollination (e.g., orchids).
Several strategies have been developed for the production of male-sterile plants (Goldberg et al., Plant Cell 5:1217-1229, 1993), including: selective destruction of the tapetum by fusing the ribonuclease gene to a tapetum-specific promoter, TA29 (Mariani et al., Nature 347:737-741, 1990); premature dissolution of the callose wall in pollen tetrads by fusing glucanase gene to tapetum-specific A9 or Osg6B promoters (Worrall et al., Plant Cell 4:759-771, 1992; Tsuchiya et al., Plant Cell Physiol. 36:487-494, 1995); antisense inhibition of flavonoid biosynthesis within tapetal cells (Van der Meer et al., Plant Cell 4:253-262, 1992); tapetal-specific expression of the Agrobacterium rhizogenes rolB gene (Spena et al., Theor. Appl. Genet. 84:520-527, 1992); and overexpression of the mitochondrial gene atp9 (Hernould et al., Proc. Natl. Acad. Sci. USA 90:2370-2374, 1993).